Basic Fibroblast Growth Factor Gene Research Articles

Transplantation of Human Embryonic Stem Cell-Derived Pericyte-Like Cells Transduced with Basic Fibroblast Growth Factor Promotes Angiogenic Recovery in Mice with Severe Chronic Hindlimb Ischemia.

Critical limb ischemia (CLI) is a state of severe peripheral artery disease, with no effective treatment. Cell therapy has been investigated as a therapeutic tool for CLI, and pericytes are promising therapeutic candidates based on their angiogenic properties. We firstly generated highly proliferative and immunosuppressive pericyte-like cells from embryonic stem (ES) cells. In order to enhance the angiogenic potential, we transduced the basic fibroblast growth factor (bFGF) gene into the pericyte-like cells and found a significant enhancement of angiogenesis in a Matrigel plug assay. Furthermore, we evaluated the bFGF-expressing pericyte-like cells in the previously established chronic hindlimb ischemia model in which bone marrow-derived MSCs were not effective. As a result, bFGF-expressing pericyte-like cells significantly improved blood flow in both laser Doppler perfusion imaging (LDPI) and dynamic contrast-enhanced MRI (DCE-MRI). These findings suggest that bFGF-expressing pericyte-like cells differentiated from ES cells may be a therapeutic candidate for CLI.

Fenugreek Seed Extract Regulates Human Umbilical Vein Endothelial Cell Angiogenesis and Proliferation via the PI3K/Akt/Cyclin D1 Pathway.

The significance of angiogenesis in tumour progression has been widely documented. Hence, the identification of anti-angiogenic agents with fewer common side effects would be valuable in cancer therapy. In this study, we evaluated the anti-angiogenic and anti-proliferative effects of a hydro-alcoholic extract of fenugreek seed (HAEF) on human umbilical vein endothelial cells (HUVECs). Human umbilical vein endothelial cells were treated with various concentrations of HAEF and the half-maximal inhibitory concentration (IC50) value was estimated by using the MTT assay. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase enzyme (MMP-2 and MMP-9) gene expression profiles were evaluated by using quantitative RT-PCR (qRT-PCR). Moreover, MMP activities and PI3K, Akt and cyclin D1 protein expression levels were evaluated by gel zymography and Western blotting, respectively. HAEF reduced HUVEC viability, with an IC50 value of 200μg/ml. The qRT-PCR results demonstrated that treatment with HAEF markedly reduced MMP-2/MMP-9, VEGF and bFGF gene expression, as compared to the control group. We also found that MMP-2/MMP-9 enzyme activity and PI3K/Akt/cyclin D1 protein expression were notably decreased in cells treated with HAEF. Our results suggest that HAEF can potentially inhibit angiogenesis, and also affect cellular proliferation by targeting the PI3K/Akt/cyclin D1 pathway. Thus, fenugreek seed extract merits further investigation as a source of compounds with anti-cancer properties.

Mesh-like electrospun membrane loaded with atorvastatin facilitates cutaneous wound healing by promoting the paracrine function of mesenchymal stem cells

BackgroundFunctional electrospun membranes are promising dressings for promoting wound healing. However, their microstructure and drug loading capacity need further improvements. It is the first time to design a novel mesh-like electrospun fiber loaded with atorvastatin (ATV) and investigated its effects on paracrine secretion by bone marrow-derived mesenchymal stem cells (BMSCs) and wound healing in vivo.MethodsWe fabricated a mesh-like electrospun membrane using a copper mesh receiver. The physical properties of the membranes were evaluated by SEM, FTIR spectroscopy, tensile strength analysis, and contrast angle test. Drug release was measured by plotting concentration as a function of time. We tested the effects of conditioned media (CM) derived from BMSCs on endothelial cell migration and angiogenesis. We used these BMSCs and performed RT-PCR and ELISA to evaluate the expressions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) genes and proteins, respectively. The involvement of FAK and AKT mechanotransduction pathways in the regulation of BMSC secretion by material surface topography was also investigated. Furthermore, we established a rat model of wound healing, applied ATV-loaded mesh-like membranes (PCL/MAT) seeded with BMSCs on wounds, and assessed their efficacy for promoting wound healing.ResultsFTIR spectroscopy revealed successful ATV loading in PCL/MAT. Compared with random electrospun fibers (PCL/R) and mesh-like electrospun fibers without drug load (PCL/M), PCL/MAT induced maximum promotion of human umbilical vein endothelial cell (HUVEC) migration. In the PCL/MAT group, the cell sheet scratches were nearly closed after 24 h. However, the cell sheet scratches remained open in other treatments at the same time point. The PCL/MAT promoted angiogenesis and led to the generation of longer tubes than the other treatments. Finally, the PCL/MAT induced maximum gene expression and protein secretion of VEGF and b-FGF. As for material surface topography effect on BMSCs, FAK and AKT signaling pathways were shown to participate in the modulation of MSC morphology and its paracrine function. In vivo, PCL/MAT seeded with BMSCs significantly accelerated healing and improved neovascularization and collagen reconstruction in the wound area compared to the other treatments.ConclusionsThe mesh-like topography of fibrous scaffolds combined with ATV release creates a unique microenvironment that promotes paracrine secretion of BMSCs, thereby accelerating wound healing. Hence, drug-loaded mesh-like electrospun membranes may be highly efficacious for wound healing and as artificial skin. It is a promising approach to solve the traumatic skin defect and accelerate recovery, which is essential to developing functional materials for future regenerative medicine.

Bioactive strontium ions/ginsenoside Rg1-incorporated biodegradable silk fibroin-gelatin scaffold promoted challenging osteoporotic bone regeneration.

Autogenous healing of osteoporotic fractures is challenging, as the regenerative capacity of bone tissues is impaired by estrogen reduction and existed pro-inflammatory cytokines. In this study, a biofunctional ginsenoside Rg1 and strontium-containing mineral (SrHPO4, SrP)-incorporated biodegradable silk fibroin-gelatin (SG) scaffold (Rg1/SrP/SG) was developed to stimulate the osteoporotic bone repair. The incorporation of 15 wt% SrP significantly enhanced the mechanical strength, stimulated the osteogenic differentiation of mouse bone marrow mesenchymal stem cells, and suppressed the osteoclastogenesis of RAW264.7 in a concentration-related manner. The loading of Rg1 in SG and 15SrP/SG scaffolds obviously promoted the angiogenesis of human umbilical vein endothelial cells via activating the expression of vascular endothelial growth factor and basic fibroblast growth factor genes and proteins. The bioactive strontium ions (Sr2+) and Rg1 released from the scaffolds together mediated lipopolysaccharide-treated macrophages polarizing into M2 type. They downregulated the expression of inflammatory-related genes (interleukin (IL)-1β, tumor necrosis factor α, and IL-6) and stimulated the expression of genes related to anti-inflammation (Arginase and IL-10) as well as bone repair (BMP-2 and PDGF-BB) in the macrophages. The in vivo results also displayed that SrP and Rg1 significantly promoted the bone repair effect of SG scaffolds in osteoporotic critical-sized calvarial defects. Besides, the degradation rate of the scaffolds was close to the bone regeneration rate. Therefore, the simultaneous addition of SrP and Rg1 is a promising way for facilitating the osteoporotic bone repair activity of SG scaffolds via promoting the osteogenesis and angiogenesis, as well as inhibiting the osteoclastogenesis and inflammation.

Molecular Evaluation of Efficacy of Freshwater and Marine Acellular Fish Skin Matrixes in Reconstruction of Ventro- Lateral Hernia in Bucks

The objective of present study is to compare between the effectiveness of freshwater and marine acellularfish skin (AFS) matrices on the reconstruction of large abdominal wall hernias in Iraqi bucks using ofmolecular evaluation depending on Real Time-quantification Polymerase Chain Reaction (RT-qPCR)technique to investigate the levels of basic-fibroblast growth factor (b-FGF) and vascular endothelial growthfactor (VEGF) genes during the healing process of the abdominal wall hernias. Ventral-lateral abdominalwall hernias (7X7cm) were induced experimentally in 18 bucks. Thirty days post-inducing of hernias, theanimals were divided randomly into two main groups (9 Bucks/ group). The hernias in animals of group (A)were treated with onlay implantation of freshwater AFS sheet. While, the hernias in animals of group (B)were treated with onlay implantation of marine AFS sheet. Molecular evaluation was depended at 2, 8, and12 weeks post-treatment of hernias. The results revealed that the significant elevation of the level of b-FGFbetween treatment groups was at week 8th in group B and at week 12th in group A. While, the significantelevation of level of VEGF between treatment groups was recorded in group A, 12 week post-treatment.

Comparative Evaluation of Viability and Function of Albino Rabbit Periodontal Ligament Fibroblasts in Various Media (In Vitro Study)

Background: Tooth avulsion is one of traumatic injuries that cause damage to periodontal ligament (PDL) tissues. Avulsed tooth should be stored in a suitable storage medium to preserve the vitality of the PDL fibroblasts (PDLFs).Aim: To assess and compare the effect of various media on albino rabbit’s PDLFs viability and basic fibroblast growth factor (b-FGF) protein and gene expressions at different time intervals.Material and methods: periodontal ligament stem cells (PDLSCs) were isolated from molars of albino rabbit then differentiated into PDLFs. The PDLFs were cultured in different media (contact lens solution, skimmed milk, Dentosafe and alcohol free mouthwash) for 1h, 12h and 24h. Phosphate buffered saline (PBS) was used as control medium. Cell viability was assessed by MTT assay. b-FGF protein and gene expression levels were detected by immunofluorescence (IF) staining and Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) respectively. Data from MTT assay and H-score of IF staining were statistically analyzed using ANOVA test and multiple two groups comparisons pos hoc test.Results: examination of isolated PDLSCs at different passages showed various shapes of cells from rounded to spindle shaped. The differentiated PDLFs showed positive IF reaction. Statistical analysis of tested media at different time intervals represented that; PBS had the highest mean of fibroblastic cell viability% and H-score, followed by Dentosafe, skimmed milk, contact lens solution and mouthwash respectively. Gene expression of b-FGF was up-regulated in Dentosafe and skimmed milk while it was down-regulated in mouthwash.Conclusions: Dentosafe is more effective in maintenance of PDLFs viability and function than the other tested media. Mouthwash is not recommended as a storage media for PDLFs.

Human mesenchymal stem cells-conditioned medium improves diabetic wound healing mainly through modulating fibroblast behaviors.

Fibroblast plays a key role in wound healing, and the advantages of mesenchymal stem cells (MSC) secretome in wound healing have previously been reported. In the present study, we investigated the impact of human bone marrow MSC-conditioned media (CM) on skin wound healing in diabetic rats and found that some improvements occurred mainly through fibroblast functions. Then, we scrutinized the impact of MSC-CM treatment on fibroblast cellular behavior by culturing human dermal fibroblasts (HDFs) in a high-glucose (HG) medium, as an in vitro diabetic model. In vivo findings revealed significant improvements in some healing kinetics of diabetic wound which received MSC-CM. Particularly, MSC-CM-treated diabetic wounds reached considerably higher percentages of wound closure. Also, the granulation tissue of these wound had less pronounced inflammatory response, better tissue remodeling, and more vascularization compared with non-treated diabetic ones. Gene expression analyses indicated that MSC-CM treatment leads to upregulation of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) genes. In addition, a significantly higher cell viability/proliferation, migration, and bFGF gene expression were observed when MSC-CM was used to treat HDFs in HG culture media. Based on these findings, it is suggested that MSC-CM could promote wound repair and skin regeneration, in some major processes, via improvement of cellular behaviors of fibroblasts in the diabetic microenvironment. The beneficial advantages of mesenchymal stem cells-conditioned media on fibroblast cellular behaviors and wound healing may lead to establish a novel approach as an alternative therapeutic procedure to cure chronic wounds in diabetic conditions.

Effect of Acellular Bovine Pericardium and Urinary Bladder Submucosa Matrixes in Reconstruction of Ventro-Lateral Hernias in Bucks; Molecular Evaluation

The present study aimed to estimate the efficiency of both a cellular bovine pericardium and bovine urinary bladder matrix sheets in the reconstruction of large ventro-lateral hernias in Iraqi bucks by using of molecular evaluation depending on real time-polymerase chain reaction technique to investigate the level of basic-fibroblast growth factor and vascular endothelial growth factor genes during the healing process and reconstruction of the abdominal defects. Under sedation and local anesthesia, (6cm X 8cm size) of ventro-lateral hernias were induced in 24 of Iraqi bucks. The animals were divided randomly into two main equal groups. In bovine pericardium-treatment group, the hernias were treated with onlay implantation of bovine pericardium. While, the hernias in UBM-treatment group were treated with onlay implantation of urinary bladder matrix, 30 days post-inducing of hernias. The molecular evaluation along the period of following-up recorded a significant up-regulation of the level of basic-fibroblast growth factor gene specific for presence of fibroblasts, myofibroblasts and collagen deposition in urinary bladder matrix -treatment group in comparison to bovine pericardium -treatment group with significant difference even at the end of the study. While, a significant up regulation of the levels of angiogenesis classic gene vascular endothelial growth factor were recorded in the bucks of bovine pericardium -treatment group compared to urinary bladder matrix -treatment group. In conclusion; molecular detection of the level of growth factors in target tissue can be used as an important criterion.

Stereological and gene expression examinations on the combined effects of photobiomodulation and curcumin on wound healing in type one diabetic rats.

We examined the effects of photobiomodulation (PBM) independently and combined with curcumin on stereological parameters and basic fibroblast growth factor (bFGF), hypoxia-inducible factor-1α (HIF-1α), and stromal cell-derived factor-1α (SDF-1α) gene expressions in an excisional wound model of rats with type one diabetes mellitus (T1DM). T1DM was induced by an injection of streptozotocin (STZ) in each of the 90 male Wistar rats. One round excision was generated in the skin on the back of each of the 108 rats. The rats were divided into six groups (n = 18 per group): control (diabetic), untreated group; vehicle (diabetic) group, which received sesame oil; PBM (diabetic) group; curcumin (diabetic) group; PBM + curcumin (diabetic) group; and a healthy control group. On days 4, 7, and 15, we conducted both stereological and quantitative real-time PCR (qRT-PCR) analyses. The PBM and PBM + curcumin groups had significantly better inflammatory response modulation in terms of macrophages (P < .01), neutrophils (P < .001), and increased fibroblast values compared with the other groups at day 4 (P < .001), day 7 (P < .01), and day 15 (P < .001). PBM treatment resulted in increased bFGF gene expression on days 4 (P < .001) and 7 (P < .001), and SDF-1α gene expression on day 4 (P < .001). The curcumin group had increased bFGF (P < .001) expression on day 4. Both the PBM and PBM + curcumin groups significantly increased wound healing by modulation of the inflammatory response, and increased fibroblast values and angiogenesis. The PBM group increased bFGF and SDF-1α according to stereological and gene expression analyses compared with the other groups. The PBM and PBM + curcumin groups significantly increased the skin injury repair process tomore rapidly reachthe proliferation phase of the wound healing in T1DM rats.

Comparative characterization of stem cells from human exfoliated deciduous teeth, dental pulp, and bone marrow–derived mesenchymal stem cells

ObjectivesMesenchymal stem cells (MSCs) are used clinically in tissue engineering and regenerative medicine. The proliferation and osteogenic differentiation potential of MSCs vary according to factors such as tissue source and cell population heterogeneity. Dental tissue has received attention as an easily accessible source of high-quality stem cells. In this study, we compared the in vitro characteristics of dental pulp stem cells from deciduous teeth (SHED), human dental pulp stem cells (hDPSCs), and human bone marrow mesenchymal stem cells (hBMSCs). Materials and methodsSEHD and hDPSCs were isolated from dental pulp and analyzed in comparison with human bone marrow (hBM)MSCs. Proliferative capacity of cultured cells was analyzed using a bromodeoxyuridine immunoassay and cell counting. Alkaline phosphatase (ALP) levels were monitored to assess osteogenic differentiation. Mineralization was evaluated by alizarin red staining. Levels of bone marker mRNA were examined by real-time PCR analysis. ResultsSHED were highly proliferative compared with hDPSCs and hBMSCs. SHED, hDPSCs, and hBMSCs exhibited dark alizarin red staining on day 21 after induction of osteogenic differentiation, and staining of hBMSCs was significantly higher than that of SHED and hDPSCs by spectrophotometry. ALP staining was stronger in hBMSCs compared with SHED and hDPSCs, and ALP activity was significantly higher in hBMSCs compared with SHED or hDPSCs. SHED showed significantly higher expression of the Runx2 and ALP genes compared with hBMSCs, based on real-time PCR analysis. In bFGF, SHED showed significantly higher expression of the basic fibroblast growth factor (bFGF) gene compared with hDPSCs and hBMSCs. ConclusionSHED exhibited higher proliferative activity and levels of bFGF and BMP-2 gene expression compared with BMMSCs and DPSCs. The ease of harvesting cells and ability to avoid invasive surgical procedures suggest that SHED may be a useful cell source for application in bone regeneration treatments.

Bioreducible Polymer Micelles Based on Acid-Degradable Poly(ethylene glycol)-poly(amino ketal) Enhance the Stromal Cell-Derived Factor-1α Gene Transfection Efficacy and Therapeutic Angiogenesis of Human Adipose-Derived Stem Cells.

Adipose-derived stem cells (ADSCs) have the potential to treat ischemic diseases. In general, ADSCs facilitate angiogenesis by secreting various pro-angiogenic growth factors. However, transplanted ADSCs have a low therapeutic efficacy in ischemic tissues due to their poor engraftment and low viability. Stromal cell-derived factor-1α (SDF-1α) improves the survival rate of stem cells transplanted into ischemic regions. In this study, we developed acid-degradable poly(ethylene glycol)-poly(amino ketal) (PEG-PAK)-based micelles for efficient intracellular delivery of SDF-1α plasmid DNA. The SDF-1α gene was successfully delivered into human ADSCs (hADSCs) using PEG-PAK micelles. Transfection of SDF-1α increased SDF-1α, vascular endothelial growth factor, and basic fibroblast growth factor gene expression and decreased apoptotic activity in hADSCs cultured under hypoxic conditions in comparison with conventional gene transfection using polyethylenimine. SDF-1α-transfected hADSCs also showed significantly increased SDF-1α and VEGF expression together with reduced apoptotic activity at 4 weeks after transplantation into mouse ischemic hindlimbs. Consequently, these cells improved angiogenesis in ischemic hindlimb regions. These PEG-PAK micelles may lead to the development of a novel therapeutic modality for ischemic diseases based on an acid-degradable polymer specialized for gene delivery.

SiRNA-mediated silencing of bFGF gene inhibits the proliferation, migration, and invasion of human pituitary adenoma cells.

Human pituitary adenoma is one of the most common intracranial tumors with an incidence as high as 16.7%. Recent evidence has hinted a relationship between growth factors of pituitary or hypothalamic origin and proliferation of human pituitary adenoma cells. This study explores the effects of small interfering RNA-mediated silencing of basic fibroblast growth factor gene on the proliferation, migration, and invasion of human pituitary adenoma cells. Human pituitary adenoma tissues were collected to obtain human pituitary adenoma cells. The basic fibroblast growth factor silencing interference plasmid was constructed, and the human pituitary adenoma cells were transfected and assigned as basic fibroblast growth factor-small interfering RNA, negative control-small interfering RNA, and blank groups. The quantitative real-time polymerase chain reaction and Western blotting were carried out to detect the expression of basic fibroblast growth factor, pituitary tumor transforming gene, vascular endothelial growth factor, cluster of differentiation 147, and matrix metalloproteinase 9. Cell Counting Kit-8 assay was conducted to observe cell proliferation at 0, 24, 48, and 72 h. Flow cytometry was used to determine cell cycle. Transwell and scratch test were applied to detect the invasion and migration of pituitary adenoma cells. Protein kinase C activity was detected. In comparison with the blank group, the basic fibroblast growth factor-small interfering RNA group showed reduced messenger RNA and protein expression of basic fibroblast growth factor, reduced cell viability at 24, 48, and 72 h, increased cells in G0/G1 stage, declined cells in S and G2/M stages, decreased number of cell migration, shortened migrating distance, reduced protein kinase C activity, and decreased expression of pituitary tumor transforming gene, vascular endothelial growth factor, cluster of differentiation 147, and matrix metalloproteinase 9. However, the negative control-small interfering RNA group had no evident differences in basic fibroblast growth factor expression, cell viability, cell cycle, number of cell migration, migrating distance, protein kinase C activity, and expression of pituitary tumor transforming gene, vascular endothelial growth factor, cluster of differentiation 147, and matrix metalloproteinase 9 compared with the blank group. The study provides evidence that small interfering RNA-mediated silencing of basic fibroblast growth factor gene inhibits the proliferation, migration, and invasion of human pituitary adenoma cells.

Basic FGF or VEGF gene therapy corrects insufficiency in the intrinsic healing capacity of tendons

Tendon injury during limb motion is common. Damaged tendons heal poorly and frequently undergo unpredictable ruptures or impaired motion due to insufficient innate healing capacity. By basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) gene therapy via adeno-associated viral type-2 (AAV2) vector to produce supernormal amount of bFGF or VEGF intrinsically in the tendon, we effectively corrected the insufficiency of the tendon healing capacity. This therapeutic approach (1) resulted in substantial amelioration of the low growth factor activity with significant increases in bFGF or VEGF from weeks 4 to 6 in the treated tendons (p < 0.05 or p < 0.01), (2) significantly promoted production of type I collagen and other extracellular molecules (p < 0.01) and accelerated cellular proliferation, and (3) significantly increased tendon strength by 68–91% from week 2 after AAV2-bFGF treatment and by 82–210% from week 3 after AAV2-VEGF compared with that of the controls (p < 0.05 or p < 0.01). Moreover, the transgene expression dissipated after healing was complete. These findings show that the gene transfers provide an optimistic solution to the insufficiencies of the intrinsic healing capacity of the tendon and offers an effective therapeutic possibility for patients with tendon disunion.

Engineering versatile protein expression systems mediated by inteins in Escherichia coli.

We have recently employed an intein, Saccharomyces cerevisiae vascular membrane ATPase (VMA), in conjunction with efficient expression and secretory functions formed between the ompA leader sequence and the human epidermal growth factor (EGF) gene (fused at the 5' end of VMA), and the human basic fibroblast growth factor (bFGF) gene (fused at the 3' end of VMA), to engineer an efficient intein-based Escherichia coli system for high-level co-expression of EGF and bFGF as authentic mature products. Both products were found not only excreted to the culture medium but also located, surprisingly, in the cytoplasm (Kwong and Wong 2013). In this study, we employed two structurally varied inteins, VMA and Mycobacterium xenopi GyraseA (GyrA), and further demonstrated that despite acting alone, both VMA and GyrA were able to mediate successful co-expression of two widely different proteins, EGF and an endoglucanase (Eng) in E. coli. Although EGF and Eng were initially expressed as large precursors/intermediates, they were soluble and auto-cleavable to finally yield the desired products in both the cytoplasm and culture media. The results further substantiate our postulation that the aforementioned intein/E. coli approach might lead to the development of cost-effective and versatile host systems, wherein all culture fractions are involved in producing the target proteins.

Repairing Injured Optic Nerve of Rat with Several Therapies

Optic nerve of mammalian, as brain and spinal cord, is difficult to regenerate successfully after injury. It is one of research focuses and difficult projects how to promote regeneration of injured optic nerve in the neuroscience and ophthalmology. We have explored to repair the injured optic nerve of rat with several therapeutic strategies in the recent years. (1)Physical therapy: In a rat model of optic nerve crush, hyperbaric oxygen preconditioning or hydrogen‐rich saline administered intraperitoneally could decrease apoptosis of retinal ganglion cells, promote their survival, and improve visual functional restoration as pupillary light reflex and visual evoked potential. (2)Cell therapy: Rat amniotic epithelial cells (AECs) with some characteristics of stem cells, AECs transfected by basic fibroblast growth factor gene, or AECs seeded in the collagen sponge was implanted into the injured region of rat optic nerve transected. These three therapies could increase the survival of retinal ganglion cells after injury of rat optic nerve. The cells could guide the regenerating axon to extend into the injured region, and promote a few of axons to cross the injured region into 1‐2 mm in the distal optic nerve. (3)Gene therapy: After neuritin lentivirus infection of rat retina, the number of retinal ganglion cells and optic nerve axons were increased significantly after optic nerve crush, this protective effects was further confirmed by the functional tests as pupillary light reflex and visual evoked potential. After RGC5 damage in vitro, neuritin overexpression promoted neurite outgrowth which might be through the upregulation of phosphorylation of Akt and Erk, and reduced cell apoptosis which might be through the inhibition of caspase 3 activation.

Positive effects of bFGF modified rat amniotic epithelial cells transplantation on transected rat optic nerve.

PurposeEffective therapy for visual loss caused by optic nerve injury or diseases has not been achieved even though the optic nerve has the regeneration potential after injury. This study was designed to modify amniotic epithelial cells (AECs) with basic fibroblast growth factor (bFGF) gene, preliminarily investigating its effect on transected optic nerve.MethodsA human bFGF gene segment was delivered into rat AECs (AECs/hbFGF) by lentiviral vector, and the gene expression was examined by RT-PCR and ELISA. The AECs/hbFGF and untransfected rat AECs were transplanted into the transected site of the rat optic nerve. At 28 days post transplantation, the survival and migration of the transplanted cells was observed by tracking labeled cells; meanwhile retinal ganglion cells (RGCs) were observed and counted by employing biotin dextran amine (BDA) and Nissl staining. Furthermore, the expression of growth associated protein 43 (GAP-43) within the injury site was examined with immunohistochemical staining.ResultsThe AECs/hbFGF was proven to express bFGF gene and secrete bFGF peptide. Both AECs/hbFGF and AECs could survive and migrate after transplantation. RGCs counting implicated that RGCs numbers of the cell transplantation groups were significantly higher than that of the control group, and the AECs/hbFGF group was significantly higher than that of the AECs group. Moreover GAP-43 integral optical density value in the control group was significantly lower than that of the cell transplantation groups, and the value in the AECs/hbFGF group was significantly higher than that of the AECs group.ConclusionsAECs modified with bFGF could reduce RGCs loss and promote expression of GAP-43 in the rat optic nerve transected model, facilitating the process of neural restoration following injury.

A single exposure to cocaine during development elicits regionally-selective changes in basal basic Fibroblast Growth Factor (FGF-2) gene expression and alters the trophic response to a second injection.

During adolescence, the brain is maturing and more sensitive to drugs of abuse that can influence its developmental trajectory. Recently, attention has been focused on basic fibroblast growth factor (FGF-2) given that its administration early in life enhances the acquisition of cocaine self-administration and sensitization at adulthood (Turner et al. (Pharmacol Biochem Behav 92:100-4, 2009), Clinton et al. (Pharmacol Biochem Behav103:6-17, 2012)). Additionally, we found that abstinence from adolescent cocaine exposure long lastingly dysregulates FGF-2 transcription (Giannotti et al. (Psychopharmacology (Berl) 225:553-60, 2013 ). The objectives of the study are to evaluate if (1) a single injection of cocaine (20 mg/kg) at postnatal day 35 alters FGF-2 messenger RNA (mRNA) levels and (2) the first injection influences the trophic response to a second injection (10 mg/kg) provided 24 h or 7 days later. We found regional differences in the FGF-2 expression pattern as either the first or the second injection of cocaine by themselves upregulated FGF-2 mRNA in the medial prefrontal cortex and nucleus accumbens while downregulating it in the hippocampus. The first injection influences the trophic response of the second. Of note, 24 h after the first injection, accumbal and hippocampal FGF-2 changes produced by cocaine in saline-pretreated rats were prevented in cocaine-pretreated rats. Conversely, in the medial prefrontal cortex and hippocampus 7 days after the first injection, the cocaine-induced FGF-2 changes were modified by the subsequent exposure to the psychostimulant. These findings show that a single cocaine injection is sufficient to produce enduring changes in the adolescent brain and indicate that early cocaine priming alters the mechanisms regulating the trophic response in a brain region-specific fashion.

Stem cells with FGF4-bFGF fused gene enhances the expression of bFGF and improves myocardial repair in rats

The aim of this study was to investigate whether the modification of bone marrow-derived mesenchymal stem cells (BMSCs) with the fused FGF4 (fibroblast growth factor 4)-bFGF (basic fibroblast growth factor) gene could improve the expression and secretion of BFGF, and increase the efficacies in repairing infarcted myocardium. We used In-Fusion technique to construct recombinant lentiviral vectors containing the individual gene of bFGF, enhanced green fluorescent protein (EGFP), or genes of FGF4-bFGF and EGFP, and then transfected these lentiviruses into rat BMSCs. We conducted an in vitro experiment to compare the secretion of bFGF in BMSCs infected by these lentiviruses and also examined their therapeutic effects in the treatment of myocardial infraction in a rodent study. Sixty rats were tested in the following five conditions: Group-SHAM received only sham operation as controls; Group-AMI received only injection of placebo PBS buffer; Group-BMSC, Group-bFGF and Group-FGF4-bFGF received implantation of BMSCs with empty lentivirus, bFGF lentivirus, and FGF4-bFGF lentivirus, respectively. Our results found out that the transplanted FGF4-bFGF BMSCs had the highest survival rate, and also the highest myocardial expression of bFGF and microvascular density as evidenced by Western blotting and immunohistochemistry, respectively. As compared to other groups, the Group-FGF4-BFGF rats had the lowest myocardial fibrotic fraction, and the highest left ventricular ejection fraction. These results suggest that the modification of BMSCs with the FGF4-bFGF fused gene can not only increase the expression of bFGF but also improve its secretion. The FGF4-bFGF BMSCs thus can enhance the survival of the transplanted cells, diminish myocardial fibrosis, promote myocardial angiogenesis, and improve cardiac functions.

Regulatory effects of introduction of an exogenous FGF2 gene on other growth factor genes in a healing tendon

In this study of a tendon injury model, we investigated how injection of a vector incorporating one growth factor gene changes expression levels of multiple growth factor genes in the healing process. The flexor tendon of chicken toes was completely cut and repaired surgically. The tendons in the experimental arm were injected with an adeno-associated virus-2 vector incorporating basic fibroblast growth-factor gene, whereas the tendons in the control arm were not injected or injected with sham vectors. Using real-time polymerase chain reaction, we found that, within the tendon healing period, a set of growth factor genes-transforming growth factor-β1, vascular endothelial growth factor, and connective tissue growth factor-were significantly up-regulated. Expression of the platelet-derived growth factor-B gene was not changed, and the insulin-like growth factor was down-regulated. A tendon marker gene, scleraxis, was significantly up-regulated in the period. Our study revealed an intriguing finding that introduction of one growth factor gene in the healing tendon modulated expression of multiple growth factor genes. We believe this study may have significant implications in determining the approach of gene therapy, and the findings substantiate that gene therapy using a single growth factor could affect multiple growth factors.

Histological and gene expression analysis of the effects of pulsed low-level laser therapy on wound healing of streptozotocin-induced diabetic rats

Diabetes mellitus (DM) is associated with poor wound healing. Studies have shown accelerated wound healing following pulsed low-level laser therapy (LLLT) in non-diabetic animals. The present study aims to evaluate the effect of pulsed LLLT on wound healing in streptozotocin-induced diabetic (STZ-D) rats. We divided 48 rats into two groups of non-diabetic and diabetic. Type 1 DM was induced in the diabetic rat group by injections of STZ. Two, full-thickness skin incisions were made on the dorsal region of each rat. One month after the STZ injection, wounds of the non-diabetic and diabetic rats were submitted to a pulsed, infrared 890-nm laser with an 80-Hz frequency and 0.2 J/cm(2) for each wound point. Control wounds did not receive LLLT. Animals were sacrificed on days 4, 7, and 15 post-injury for histomorphometry and reverse transcription polymerase chain reaction (RT-PCR) analyses of basic fibroblast growth factor (bFGF) gene expression. Pulsed LLLT significantly increased the numbers of macrophages, fibroblasts, and blood vessel sections compared to the corresponding control groups. Semi-quantitative analysis of bFGF gene expression at 48 h post-injury revealed a significant increase in gene expression in both non-diabetic and diabetic rats following LLLT (the ANOVA test). Pulsed LLLT at 0.2 J/cm(2) accelerated the wound healing process in both non-diabetic and diabetic rats as measured by histological characteristics and semi-quantitative bFGF gene expression.